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Clin Vaccine Immunol. 2010 January; 17(1): 98–107.
Published online 2009 November 18. doi:  10.1128/CVI.00342-09
PMCID: PMC2812078

Evaluation of a Whole-Blood Cytokine Release Assay for Use in Measuring Endotoxin Activity of Group B Neisseria meningitidis Vaccines Made from Lipid A Acylation Mutants [down-pointing small open triangle]

Abstract

Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-α assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.

Endotoxin, or lipopolysaccharide (LPS), which is found in the outer membrane of Gram-negative bacteria, is a key virulence factor for many Gram-negative bacteria. Neisseria meningitidis, which is an important etiological agent of bacterial meningitis, is an organism that produces and sheds a potent endotoxin often referred to as lipooligosaccharide (LOS) because it has no O side chain. In this paper, however, we refer to it in the traditional manner as LPS. The presence of LPS in drugs and biologicals intended for human use has long been recognized as a marker for bacterial contamination and a potential cause of adverse reactions (47). The rabbit pyrogen test (RPT) was established in the 1940s as a standard requirement for end product safety testing (27). However, much of routine end product testing previously done with the RPT was replaced by the subsequently developed Limulus amebocyte lysate (LAL) in vitro assay for LPS (9, 10).

Currently, an increasing number of LPS-related molecules are being investigated for use for the treatment of sepsis (20) or for use as vaccines or vaccine adjuvants (13, 14, 30). One group of vaccine candidates that includes LPS as a major component is based on native outer membrane vesicles (NOMVs) from group B N. meningitidis. These experimental vaccines consist of purified outer membrane vesicles or blebs produced by the meningococci during normal growth and have been used both intranasally and parenterally (7, 26). Genetically detoxified N. meningitidis strains that express normal amounts of LPS with reduced toxicity have been developed. The LPS is a desirable component of meningococcal NOMV vaccines because it has been shown to induce bactericidal antibodies, is relatively conserved, and can have significant adjuvant activity (6, 12, 16, 41). The genetic mutations that were used to reduce the endotoxin activity of the LPS are analogous to those first introduced into Escherichia coli by disabling the htrB or msbB and code for the acyltransferases that attach the secondary acyloxyacyl-linked fatty acids to the lipid A of the LPS (3). These two genes were later renamed lpxL and lpxM, respectively, because of the important role that they play in LPS biosynthesis (4, 35). Analogous mutations were first introduced into N. meningitidis by van der Ley et al., who labeled the corresponding genes lpxL1 and lpxL2. LpxL1 and lpxL2 knockouts result in strains that synthesize variant LPS molecules containing penta-acyl or tetra-acyl lipid A, respectively, rather than the wild type hexa-acyl lipid A (44).

While the RPT and the LAL assay are sensitive to many forms of endotoxin, the ability of these assays to mimic the human response to wild-type and modified endotoxins from different sources has not been thoroughly evaluated. Indeed, since these tests are based on the recognition of endotoxin by nonhuman systems, they are unlikely to mimic exactly the human response to the same molecules. It has been shown that the Toll-like receptor 4 (TLR4) molecules of different species respond differently to various structural variants of LPS (15, 17, 40). Therefore, there is a need for assay methods that can reliably predict the safety of vaccines and other biologicals designed for human use that contain modified endotoxins. One group of assays under consideration for use in estimating the potencies of vaccines and other biologicals are in vitro cytokine release assays with human whole blood (WB) or isolated peripheral blood mononuclear cells (PBMCs). Because these assays rely on the responses generated by human cells, the potential to more accurately estimate the human response with these systems rather than the LAL assay or RPT exists. Fennrich et al., using 10 different endotoxins from various Gram-negative bacteria, demonstrated that a whole-blood cytokine release assay positively correlated with the in vivo endotoxicity of the different LPS structures in rabbits (11). They found the cytokine release assay to be superior to the RPT and the LAL assay for the discrimination of various LPS molecules. The sensitivities and specificities of various cytokine release assays in detecting endotoxin standards have been defined (22), and several human monocyte-based in vitro pyrogen tests have been validated for use for the detection of endotoxin and Gram-positive bacterial pyrogens (21).

Here we report on the ability of whole-blood interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) release assays to discriminate wild-type hexa-acyl Neisseria LPS from the penta-acyl ΔlpxL1 and tetra-acyl ΔlpxL2 variants used in NOMV vaccines. A method for expressing the endotoxin potency of a test sample as the mean pg of cytokine released per ng of stimulating antigen is also presented. Both the IL-6 and the TNF-α release assays with whole blood but not the IL-6 and TNF-α release assays with PBMCs were able to discriminate between NOMV vaccines containing penta-acyl and tetra-acyl LPSs, suggesting that the in vitro assay with human whole blood may be more sensitive than the assay with PBMCs to variations in the structure of lipid A. Once it is correlated with the clinical outcome, the assay with human whole blood has the potential to play a significant role in the development and regulation of vaccines and other biologicals that contain variant lipid A structures.

MATERIALS AND METHODS

Preparation of vaccines and purified LPS.

Clinical lots of NOMV vaccines were prepared from genetically modified vaccine strains by extraction methods that have been described previously (36). Briefly, NOMVs were extracted from packed bacterial cells by suspending the bacterial cells in buffer containing 0.15 M NaCl, 0.05 M Tris-HCl, and 0.01 M EDTA (pH 7.5); incubating the mixture at 56°C for 30 min; and shearing the cells in a Waring blender for 3 min. The cells were pelleted, resuspended in 0.01 M Tris-HCl, pH 7.5, and again sheared in the blender. The cells and the cell debris were removed by centrifugation, and the two supernatants were combined. The NOMVs were twice pelleted by ultracentrifugation, and the pellet was resuspended in 0.01 M Tris-HCl (pH 7.5). The protein content was determined by the method of Lowry et al. (29), and the LPS content was determined by the 2-keto-3-deoxyoctonate method of Karkhanis et al. (25). Table Table11 gives a brief summary of the compositions of the three NOMV vaccines used in this study: the 44/76 M2 NOMV vaccine containing hexa-acyl LPS, the 8570 HOPS-G ΔlpxL1 NOMV vaccine containing penta-acyl LPS, and the 44/76 MOS 5D ΔlpxL2 NOMV vaccine containing tetra-acyl LPS.

TABLE 1.
Protein and LPS contents of clinical lots of three NOMV vaccines

Preparation of purified N. meningitidis LPS.

Hexa-acyl, penta-acyl, and tetra-acyl LPSs were extracted from capsule-negative strains of N. meningitidis by the hot phenol-water extraction method of Westphal and Jann (46). All the LPSs obtained except for the LPS extracted from the ΔlpxL2 mutant strain were further purified, as described previously (12). The LPS extracted from the ΔlpxL2 mutant did not pellet in the ultracentrifuge and thus required a different method for final purification. Following the removal of phenol by dialysis against water for 48 h, the ΔlpxL2 LPS was dialyzed against buffer containing 0.05 M Tris (pH 8.0), 0.15 M NaCl, 1 mM EDTA (pH 8.0), and 0.8% Empigen BB detergent. The nucleic acid was removed by batch adsorption onto DEAE-cellulose DE-52 (Whatman, Inc., Clifton, NJ). For every 5 ml of LPS extract, 1.5 g of DE-52 was added. The suspension was mixed for 30 min and filtered through Whatman filter paper. Low-molecular-weight molecules were removed by ultrafiltration with a UFP-3-C-MB column (A/G Technology Corp., Needham, MA) and washing with about 10 volumes of water. The absence of nucleotide contamination was determined by a lack of UV absorbance at 260 nm.

Other purified reagents.

Purified hexa-acyl E. coli O111:B4 LPS was purchased from Lonza (product no. L5293). Monophosphoryl lipid A (MPLA) from E. coli F583 was purchased from Sigma-Aldrich (product no. L6638).

LAL assay.

The LAL assay (catalog no. G5006; Pyrotell Associates, Cape Cod, MA) for determination of endotoxin activity was used according to the manufacturer's instructions. Complete clotting of the lysate was indicative of a positive reaction.

RPT.

Vaccines were tested for pyrogenicity in the standard RPT in accordance with 21 CFR 610.13. The assays were done under contract with BioReliance (Rockville, MD). Briefly, three 7-week-old New Zealand White rabbits were maintained under closely monitored conditions and were injected with approximately 3 ml/kg of body weight of an appropriate dilution of the vaccine in question. Rectal temperatures were measured at half-hour intervals. A sample was considered nonpyrogenic if no rabbit showed a rise in body temperature of 0.5°C or more above its baseline temperature for 1 to 3 h postinoculation. Pyrogen tests were performed over a range of concentrations of NOMV containing hexa-acyl, penta-acyl, and tetra-acyl LPSs to find the highest concentration that did not induce a fever.

Stimulation of human WB with vaccines or purified LPS.

The WB cytokine release assay was performed as described in Current Protocols in Immunology (43). The release of IL-6 and TNF-α was quantified because pilot studies indicated that human WB quickly responds to stimulation with meningococcal vaccines and endotoxins by releasing large quantities of these two proinflammatory cytokines. Preliminary studies with WB from four donors indicated that the proinflammatory reactions to meningococcal vaccines were fairly consistent from donor to donor both in magnitude and in the ranking of the responses to different antigenic preparations. Studies have shown that genetic variations in genes coding for TLR4, TNF, and CD14 may indeed alter the in vitro inflammatory responses of tissues (23); however, in order to eliminate as much variation in the model system as possible, fresh whole blood was exclusively obtained from a single arbitrarily chosen donor. Blood was collected in heparinized tubes according to an approved human use protocol and was kept at room temperature for 4 h. Heparin was considered the best available anticoagulant for this study because, unlike other widely used anticoagulants, such as EDTA and warfarin, heparin is endogenously found in human whole blood. A 245-μl sample of blood was dispensed into each well of a 96-well tissue culture plate. Serial dilutions of each vaccine or antigen were prepared in RPMI 1640 cell culture medium (catalog no. 72400-047; Gibco). Eight fourfold dilutions of the 44/76 M2 NOMV vaccine were prepared such that the concentration (protein) of vaccine in the eight wells ranged from 1.6 × 103 ng/ml to 0.098 ng/ml. Nine fourfold dilutions of the 8570 HOPS-G NOMV and 44/76 MOS 5D NOMV vaccines were prepared such that the final concentration (protein) of vaccine in the nine wells ranged from 4.0 × 103 ng/ml to 0.61 ng/ml. Eight 10-fold dilutions of both purified hexa-acyl E. coli O111:B4 LPS and purified hexa-acyl N. meningitidis LPS were prepared such that the final concentration (LPS, dry weight) of vaccine in the eight wells ranged from 4.0 × 103 ng/ml to 0.4 × 10−3 ng/ml. Eight 10-fold dilutions of both purified MPLA from E. coli F583 and purified penta-acyl N. meningitidis LPS were prepared such that the concentration (dry weight) of vaccine in the eight wells ranged from 1.0 × 105 ng/ml to 0.010 ng/ml. Eight 10-fold dilutions of purified tetra-acyl N. meningitidis LPS were prepared such that the final concentration (dry weight) of LPS in the eight wells ranged from 1.0 × 106 ng/ml to 0.1 ng/ml. Five microliters of each diluted sample was dispensed into each assigned well to reach a final volume of 250 μl. The whole blood was incubated for 4 h at 37°C with 5% CO2 and was then briefly centrifuged. A sample of 55 μl of plasma from each well was removed and frozen pending quantification of the cytokines.

Stimulation of human CD14+ PBMCs with vaccines or purified LPS.

Cryopreserved, untreated CD14+ peripheral blood monocytes (series no. 2W-400; Lonza, Basel, Switzerland) were thawed and suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 0.05 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 50 U/ml penicillin, and 50 μg/ml streptomycin. Macrophage colony-stimulating factor (M-CSF) was added to a concentration of 250 ng/ml, and the cells were incubated overnight. The cells were transferred to 96-well plates at a concentration of 2 × 105 cells per well. The vaccines were prepared in RPMI 1640 cell culture medium. Five fourfold dilutions of the 44/76 M2 NOMV vaccine were prepared such that the concentration (protein) of vaccine in the five wells was in the range of 10 ng/ml to 0.039 ng/ml. Eight fourfold dilutions of the 8570 HOPS-G NOMV and 44/76 MOS 5D NOMV vaccines were prepared such that the concentration (protein) of vaccine in the eight wells was in the range of 1.0 × 103 ng/ml to 0.061 ng/ml. Five microliters of each dilution or 5 μl of a negative control consisting of only RPMI 1640 medium was dispensed into each assigned well, and the plates were incubated for 4 h at 37°C with 5% CO2. Following incubation, the cells were centrifuged and the supernatant was stored at −80°C.

Quantification of proinflammatory cytokine release.

The supernatants were assayed for IL-6 and TNF-α with a human cytokine LINCOplex Hcyto-60K kit (Millipore, Billerica, MA), according to the manufacturer's instructions. The method used allowed for a range of sensitivities from 1.0 pg/ml to 15,600 pg/ml of IL-6 or TNF-α. Unknown samples were dispensed into 96-well filter plates. Latex beads coated with monoclonal antibodies for IL-6 and TNF-α were added to each well, followed by the addition of a biotin-labeled secondary antibody and a streptavidin-phycoerythrin conjugate. The plate was processed with a Luminex100 IS system (Luminex Corporation, Austin, TX). The resulting median fluorescence intensity (MFI) data points produced as the output from the Luminex100 system were analyzed by the Upstate BeadView (version 1.0) statistical software program from Millipore, according to the developer's instructions. These data were reported as pg of cytokine detected per ml of sample.

Analysis of proinflammatory cytokine release data.

Pilot experiments were performed to determine at which regions of the cytokine response curve the release of cytokine from whole blood and PBMCs was linearly related to the concentration of the stimulating antigen. Whole-blood samples showed a linear or a nearly linear relationship in a range of 100 to 8,000 pg TNF-α and 75 to 6,000 pg IL-6, respectively, whereas PBMCs showed the most linear response in the range of 30 to 2,500 pg IL-6 and 25 to 2,000 pg TNF-α.

Endotoxin activity was calculated as the geometric mean of the ratios of the cytokine concentration (in pg/ml) to the antigen concentration (in ng/ml) for points falling within the linear portion of the response curve. The Kruskal-Wallis nonparametric analysis of variance (ANOVA) was used to determine if there were differences among the different vaccines or pyrogens being tested. When the ANOVA result was statistically significant (P < 0.05), Mann-Whitney U tests were used to determine where the differences lay. Finally, the geometric mean of the mean ratios previously calculated was calculated to obtain a mean potency over several assays. The result was a single value with units given as the mean pg of cytokine released per nanogram of stimulatory antigen.

RESULTS

LAL assay.

The endotoxin activities of the wild-type 44/76 M2 NOMV vaccine containing the hexa-acyl LPS, the 8570 HOPS-G ΔlpxL1 NOMV vaccine containing the penta-acyl LPS, and the 44/76 MOS 5D ΔlpxL2 NOMV vaccine containing the tetra-acyl LPS were quantified by the LAL assay and are reported in Table Table2.2. A comparison of the relative activities of the vaccines revealed that there was a minimal difference between the endotoxic activity of the wild-type vaccine and the activities of the ΔlpxL1 and ΔlpxL2 NOMV vaccines. The LAL test showed all acylation variants to be highly active and to have activities within the same order of magnitude. Thus, the LAL assay was insensitive to the acylation state of the lipid A and is thus not useful for predicting the endotoxin activity in humans of LPS with modified lipid A acylation.

TABLE 2.
Toxicities of NOMV vaccines containing hexa-acyl, penta-acyl, and tetra-acyl LPSs, as determined with an LAL assay system and RPT

Rabbit pyrogenicity.

In the RPT, the highest nonpyrogenic dosages of the wild-type 44/76 M2 NOMV vaccine containing the hexa-acyl LPS, the 8570 HOPS-G ΔlpxL1 NOMV vaccine containing the penta-acyl LPS, and the 44/76 MOS 5D ΔlpxL2 NOMV vaccine containing the tetra-acyl LPS were determined and are reported in Table Table2.2. A comparison of the highest nonpyrogenic dose for each vaccine in the RPT indicated that the ΔlpxL1 NOMV vaccine was about 40-fold less pyrogenic than the wild-type NOMV vaccine and that the ΔlpxL2 NOMV vaccine was about 200-fold less pyrogenic than the wild-type vaccine. Additional RPTs done with several different laboratory preparations of NOMVs containing ΔlpxL1 LPS gave results that varied from being from 10- to 40-fold less pyrogenic than the NOMV vaccine containing the wild-type LPS (data not shown).

In vitro stimulation of human WB with vaccines.

The NOMV vaccines with the wild-type, ΔlpxL1, and ΔlpxL2 LPSs were examined by the human WB assay. The averaged cytokine release data from seven such experiments are plotted against the concentration of stimulating antigen in Fig. Fig.1A1A for IL-6 and Fig. Fig.1B1B for TNF-α. The results of further analysis of the data by the statistical method outlined in the Materials and Methods section are presented in Table Table33 as the mean endotoxin activity (pg cytokine released/ng of stimulating antigen) for IL-6 and TNF-α release for each vaccine. The NOMV vaccine containing the hexa-acyl LPS was found to be significantly more active than the vaccines containing either the penta-acyl or the tetra-acyl LPS. The assay with human WB was also able to detect greater cytokine stimulating activity for the vaccine containing the penta-acyl LPS than the vaccine containing the tetra-acyl LPS. No difference in cytokine stimulation by the vaccines containing the penta-acyl and the tetra-acyl LPSs was observed in the assay with PBMCs. The results of the statistical analyses of the results are summarized in Table Table44.

FIG. 1.
Average results of seven in vitro whole-blood stimulation assays comparing the responses to vaccines comprising NOMVs prepared from strains expressing LPSs with different lipid A structures. The error bars represent the standard errors of the means.
TABLE 3.
IL-6 and TNF-α release following stimulation of whole blood by LPS-containing vaccines, as measured by the cytokine release assay
TABLE 4.
Summary of statistical analysis of the geometric mean ratios of picograms of cytokine released per nanogram of test sample calculated from data lying in the linear response region of the cytokine release curves

In vitro stimulation of human WB with purified LPS.

To further analyze the relative toxicities of the LPS constituents of the vaccines independently of the other NOMV constituents, human WB cytokine release assays were performed with purified meningococcal hexa-acyl, penta-acyl, and tetra-acyl LPSs as well as MPLA and purified hexa-acyl E. coli O111:B4 LPS as a positive control. These data are presented in Fig. 2A and B.

FIG. 2.
Results from one of several similar in vitro whole-blood stimulation assays comparing the responses to purified LPS molecules from strains expressing LPSs with different lipid A structures.

Stimulation of human PBMCs with vaccines.

The mean data from seven PBMC stimulation experiments are presented in Fig. 3A and B as cytokine release curves over a range of stimulating antigen concentrations. The results of further analysis of the IL-6 and TNF-α data based on the statistical method outlined in the Materials and Methods section are presented in Table Table5.5. Like the data from the in vitro stimulation of WB by vaccines, the data were analyzed to assess the assay's ability to detect differences in the responses to the wild-type, ΔlpxL1, and ΔlpxL2 vaccines. As summarized in Table Table4,4, the results of the PBMC assay showed significant differences in the amounts of IL-6 and TNF-α released when PBMCs were stimulated with vaccine containing hexa-acylated LPS than when they were stimulated with vaccine containing penta-acylated or tetra-acylated LPS. However, the PBMC system was not able to detect a significant difference between the vaccines containing ΔlpxL1 and ΔlpxL2 LPS molecules.

FIG. 3.
Average results of seven in vitro human peripheral blood monocyte stimulation assays comparing the responses to vaccines comprising NOMVs prepared from strains expressing LPSs with different lipid A structures. The error bars represent the standard errors ...
TABLE 5.
IL-6 and TNF-α release following stimulation of PBMCs by LPS-containing vaccines, as measured by the cytokine release assay

DISCUSSION

We have shown that a whole-blood cytokine release assay is capable of discriminating between wild-type N. meningitidis LPS and LPS from mutant strains that express penta- or tetra-acylated lipid A. When the LAL assay was used to evaluate the same vaccines, it was insensitive to changes in lipid A acylation, and the RPT was less sensitive to these variations in lipid A acylation, particularly the difference between the NOMV vaccines containing the wild-type and the penta-acyl LPSs. In the whole-blood IL-6 and TNF-α assays, the NOMV vaccine containing either the penta-acyl or the tetra-acyl LPS appeared to be highly detoxified compared to the level of detoxification for the NOMV vaccine containing the wild-type hexa-acyl NOMV. Furthermore, the WB assay could distinguish between these acylation variants, whereas the PBMC assay could not. The difference revealed by the WB assay suggests that meningococcal LPS containing tetra-acylated lipid A is less able to stimulate the production of proinflammatory cytokines in the human system than penta-acylated LPS.

Although our data are consistent with the current understanding of the TLR4-dependent inflammatory response, other factors besides the number of acyl chains could potentially alter the potencies of the preparations tested and must also be considered. First, the concentrations of NOMV preparations used in our study were based on the protein rather than the LPS content, and the ΔlpxL2 vaccine contained less LPS relative to protein than the ΔlpxL1 or wild-type NOMV vaccine (Table (Table1).1). Second, it has been suggested that the lpxL mutations may result in an altered vesicle morphology. such that the LPS binding in the vesicle or its exposure at the surface is altered (12). The morphology of the NOMV and the strength with which LPS is bound to the vesicle can have a profound impact on cytokine release characteristics, as demonstrated by Bjerre et al. (1). Alterations in the solubility of LPS, as well as its ability to bind to human MD-2 and TLR4, are other potential sources of variation in endotoxin potency.

While the other factors mentioned above have been shown to alter the potency of bacterial endotoxins, our results are consistent with the theory that the TLR4-dependent responses observed were generally dependent on the type of LPS present in the vesicles. The N. meningitidis NOMV vaccine containing tetra-acylated ΔlpxL2 LPS induced slightly less IL-6 and TNF-α in whole blood than the ΔlpxL1 NOMV vaccine containing the penta-acylated LPS, and NOMV vaccines containing either tetra- or penta-acylated LPS were much less potent than NOMV vaccines containing hexa-acylated LPS. The challenge of whole blood with purified meningococcal hexa-, penta-, and tetra-acyl LPSs gave results similar to those obtained by challenge with NOMVs containing hexa-, penta-, or tetra-acyl LPS. The similar activities of the purified penta- and tetra-acyl LPSs suggest that when they are in the vesicle, other vesicle components, such as the outer membrane proteins, may be adding to the overall stimulatory activity observed for the ΔlpxL2 vesicles (24). Since the ΔlpxL2 NOMV vaccine contained only one-sixth as much LPS as the penta-acyl NOMV vaccine, one might have expected to see a greater difference between the LPSs in the ΔlpxL2 NOMV and the ΔlpxL1 NOMV vaccines. Other studies involving tetra-acyl LPS or lipid A and lipid A-like molecules have indicated that these molecules appear to be nonpyrogenic or antipyrogenic (14, 15, 28). Investigators have previously demonstrated that the lipid A from neisserial ΔlpxL1 and ΔlpxL2 mutants binds to human MD-2 and TLR4 because they have been used as competitive inhibitors of hexa-acyl lipid A (2, 40, 42). Since meningococcal outer membrane proteins can also stimulate cytokine release (24, 39), protein-mediated stimulation may account for a certain degree of background IL-6 and TNF-α release apparent in the cytokine release assays involving NOMV vaccines and may thereby partially mask the true difference in endotoxin activity between the ΔlpxL1 and the ΔlpxL2 LPSs present in the vesicles.

Both the 8570 HOPS-G ΔlpxL1 NOMV vaccine (lot 1289; penta-acyl LPS) and the 44/76 MOS ΔlpxL2 NOMV 5D vaccine (lot 0832; tetra-acyl LPS) have been found to be well tolerated in human phase I clinical trials when they were given intramuscularly to 34 and 36 volunteers, respectively (unpublished data and reference 26). None of the doses of the 44/76 MOS ΔlpxL2 NOMV 5D vaccine given exceeded the threshold of the μg vaccine (by protein)/kg reached in the RPT of 2 μg/kg. However, the 8570 HOPS-G ΔlpxL1 NOMV vaccine was found to be pyrogenic in rabbits at a dose above 35 μg (0.4 μg/kg) and was given in a dose-escalating fashion up to a dose of 75 μg, or about 1 μg/kg. Although clinically imperceptible increases in temperature did occur postvaccination, no fevers were measured or reported, including after the administration of the highest dosages of 50 μg (nine volunteers, three doses) and 75 μg (nine volunteers, three doses). Dosage-dependent increases in the white blood cell counts were not detected in either study. Cytokine release tests with the 8570 HOPS-G ΔlpxL1 NOMV vaccine showed a much greater fold reduction in endotoxin activity than that determined by the RPT. The very mild responses of volunteers to even the highest vaccine doses strongly suggest that the cytokine assay more accurately predicts the effects of the modified endotoxin in humans than the RPT.

Although the use of a human monocyte cell line or PBMCs may be attractive from the standpoint of assay standardization, there are several reasons we believe that the use of human whole blood for the cytokine release assay is preferable. It is perhaps intuitive that whole blood better models the complex interplay of multiple cell types found in vivo (5). The use of whole blood eliminates the potential stress and storage time associated with purification of the monocytes. Thus, their response to stimulation is more likely to mimic an in vivo response. In one study, purified monocytes were found to be more sensitive to endotoxin than whole blood on the basis of the release of IL-8 (28), but this difference was slight. In a comparison of in vitro cytokine release tests done by the Interagency Coordinating Committee on the Validation of Alternative Methods, the assays performed with whole blood had slightly lower sensitivities than the assays performed with cultured cell lines (22), but the difference reported was small (89% versus 96%) and was based only on hexa-acylated E. coli endotoxin standards. Monocytes are often considered to be one of the most important populations of blood cells for the recognition of bacterial endotoxin and the subsequent inflammatory reaction (5, 31, 33, 48). Indeed, the fact that the monocyte cytokine release assay clearly distinguishes the wild-type LPS from the two mutant forms suggests that this test may be a good test for screening for the presence of wild-type LPS due either to contamination or to reversion. However, in our studies the assay with whole blood was found to be more sensitive to variations in lipid A acylation than assays that utilized isolated PBMCs.

The differences observed between the PBMC and the whole-blood assays could be explained in part by several important differences in these systems. First, artifacts may have resulted from the purification, cryopreservation, and thawing of the PBMCs. A second possible factor stems from the use of heparin as an anticoagulant in the whole-blood assay. Heparin has been shown in some in vitro studies to increase the sensitivity of whole blood to endotoxin stimulation (19) and therefore may have contributed in some way to the variations observed in the two assay systems. Finally, whole-blood samples contain white blood cell populations, such as granulocytes and neutrophils (18, 45), and various other blood components, including lipopolysaccharide-binding protein, soluble CD14, thromboxane, serum lipoproteins, and platelet-activating factor (8, 32, 34, 37, 38, 42), that have been shown to interact with LPS or play a role in the production of immunologically active cytokines.

In the more than 6 decades since the RPT was codified into the U.S. Pharmacopoeia and in the more than 4 decades since the LAL assay was developed, our appreciation of the potential for structural variations in lipid A has grown. Along with it, our understanding of innate immunity and the TLR recognition system has greatly increased, and the potential now exists for the inclusion of variants of lipid A in products developed to prevent and treat disease. This emerging class of biologicals will need a reliable in vitro test to predict their safety and efficacy. The high degree of sensitivity of the WB assay to variations in lipid A structure and the relevance of using human cells to predict safety in humans suggests that the whole-blood cytokine release test might be considered the test of choice for use in the development of vaccines and other biologicals for human use, particularly if modified lipid A structures are present.

Acknowledgments

We thank Elizabeth Moran, Joseph Labrie, Deborah Schmiel, Romanza Green, Robert Burden, Brenda Brandt, and others involved with the Meningococcal Vaccine Development of the Department of Bacterial and Rickettsial Diseases for their support. We also thank Andy LaClair for his assistance with the Luminex instrument, Naomi Fineberg for her assistance with the biostatistics, and the staff of the WRAIR Pilot Bioproduction Facility for manufacturing and performing the LAL assays with the NOMV vaccines.

This study was funded by the U.S. Army Medical Research Materiel Command through its Military Infectious Disease Research Program.

The manuscript has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the true views of the U.S. Department of the Army or the U.S. Department of Defense. This research was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to the principles stated in the Guide for the Care and Use of Laboratory Animals (33a).

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 November 2009.

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